Phytochrome regulated transcription factor for control of higher plant development

ABSTRACT

The present invention involves the isolation and characterization of the first discovered phytochrome-regulated transcriptional factor, a protein designated CCA1 which binds to the promoter region of the chlorophyll binding protein gene (Lhcb1*3) of Arabidopsis. The Lhcb1*3 gene of Arabidopsis is known to be regulated by phytochrome in etiolated seedlings where a brief illumination by red light results in a large increase in the level of mRNA from this gene. A DNA binding activity, designated CA-1, that interacts with the promoter region of Lhcb1*3 was previously discovered in cellular extracts. This binding activity was used to obtain a cDNA clone for a transcription factor that binds specifically to the Lhcb1*3 promoter. Modification of the expression of CCA1 using techniques of genetic engineering results in unexpected changes in the timing of plant flowering. When CCA1 is overexpressed, it appears that the normal circadian rhythms of the plant are disrupted. The plants take a significantly longer time to reach flowering even in the presence of day length conditions that normally induce flowering. Thus, a method of extending vegetative growth and delaying flowering is provided.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention concerns the field of genetic engineeringand more particularly the discovery of a unique light-regulatedtranscription factor that can be used to control the flowering time ofplants.

[0003] 2. Background of the Invention

[0004] The Sun is the primary source of energy on the Earth. It isobvious that impinging solar energy warms our atmosphere and drives theEarth's climate. Perhaps less obvious is that virtually all biologicalenergy including the “fossil” fuels that power our civilization aresolar in origin. Solar energy is captured for biological use byphotosynthesis, a metabolic process that occurs in green plants. Duringphotosynthesis light energy is captured in various chemical compoundsthat provide food for all nonphotosynthetic organisms.

[0005] Since green plants essentially “feed” on light, it comes as nosurprise that these organisms are exquisitely sensitive to light. Manypeople are aware that plants grow towards a light source in an effort toreceive sustaining illumination. However, a green plant's responsivenessto light is much more complex than merely growing towards a lightsource. Plants contain complex systems for actually measuring theduration of day and night lengths so as to synchronize their growth andlifecycles with the seasons. It is these timing processes that causechrysanthemums to flower in the autumn and other ornamental and cropplants to flower and fruit at characteristic times. Clearly, the abilityto accurately control flowering to promote it or delay it as necessarywould be of great economic value. In the middle decades of this centurya tremendous amount of biological research was carried out in search ofthe ever elusive “flowering hormone” or florigen which, for a time, wasthe holy grail of plant physiology. Although the quest for florigenended in failure, much was learned about how plants perceive and respondto environmental factors such as the seasonal changes in day length.

[0006] Although green plants have multiple light receptors, theprotein-pigment phytochrome has been shown to be the primary receptor bywhich plants track day length and orchestrate a number oflight-regulated responses. Phytochrome is a chromoprotein formed bycombining a linear tetrapyrolle pigment with an apoprotein. As such itshows some similarities to phycobiliproteins which are accessorypigments of certain algae and photosynthetic bacteria. Phytochrome hasthe somewhat unusual property of existing in two differentphotochemically interconvertible forms know as Pr (phytochrome-red) andPfr (Phytochrome-far red). Phytochrome is synthesized in the Pr formwhich has an absorption maximum in the red region of the opticalspectrum. Numerous experiments have shown that the Pr form ofphytochrome is essentially inactive in terms of eliciting changes inplant metabolism. However, when Pr absorbs red light (R), it is rapidlyconverted into the active Pfr form. Pfr has an absorption maximum in thefar red (near infrared) portion of the optical spectrum. Absorption offar red light (FR) induces a back conversion of Pfr to inactive Pr. Thisred-far/red interaction provides a powerful test of whether a givenplant response is phytochrome mediated. For example, if dark-grown(etiolated) seedlings are briefly exposed to red light, Pfr will beformed and there will be a concomitant response. However, if the redlight exposure is quickly followed by a far-red light exposure (whichconverts Pfr to inactive Pr) the response will be prevented. Thereversibility of a red light response by a far-red light exposure is ahallmark of a phytochrome response.

[0007] Although much is known about the phytochrome proteins and theirencoding genes, relatively little is known about how the Pfr effects aresponse in the plant. Many plant genes are light-regulated and that atleast some of this regulation is controlled or influenced byphytochrome. Among the genes whose expression is either negatively orpositively influenced by phytochrome are several that have been shown tobe transcriptionally regulated. These genes include those encoding thesmall subunit of ribulose bisphosphate carboxylase/oxygenase, the majorlight-harvesting chlorophyll a/b binding-proteins (Lhcb) of PhotosystemII, NADPH: protochlorophyllide oxioreductase, ferredoxin andphosphoenolpyruvate carboxylase, all components of photosynthesis. Whilethe promoter regions are known for many of these genes, thetranscriptional factors that bind to these nucleic acid regions aregenerally unknown. Furthermore, the signal transduction pathwaysconnecting Pfr to these transcriptional factors are largely unknown.These matters have been recently reviewed in Tobin, E. M. and Kehoe D.M. “Phytochrome regulated gene expression,” Seminars in Cell Biology 5:335-46 (1994) to which the reader is directed for more detailedinformation.

SUMMARY OF THE INVENTION

[0008] The present invention involves the isolation and characterizationof the first discovered phytochrome-regulated transcriptional factor, aprotein designated CCA1 which binds to the promoter region of achlorophyll binding protein gene (Lhcb1*3) of Arabidopsis. The Lhcb1*3gene of Arabidopsis is known to be regulated by phytochrome in etiolatedseedlings where a brief illumination by red light results in a largeincrease in the level of mRNA from this gene. Karlin-Neumann, G. A.,Sun, L., and Tobin, E. M. Plant Physiol. 88:1323-31 (1988). A DNAbinding activity, designated CA-1, that interacts with the promoterregion of Lhcb1*3 was discovered in cellular extracts. Sun, L., Doxsee,R. A., Harel, E., and Tobin, E. M., Plant Cell 5: 109-21 (1993) (Sun etal., 1993). The promoter region to which CA-1 binds has been shown to benecessary for normal phytochrome regulation of the Lhcb1*3 gene.Kenigsbuch, D. and Tobin, E. M. Plant Physiol. 108:1023-27 (1995).Modification of the expression of CCA1 using techniques of geneticengineering results in unexpected changes in the timing of plantflowering.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The objects and features of the present invention, which arebelieved to be novel, are set forth with particularity in the appendedclaims. The present invention, both as to its organization and manner ofoperation, together with further objects and advantages, may best beunderstood by reference to the following description, taken inconnection with the accompanying drawings.

[0010]FIG. 1 gives sequences of the A2 fragment of the Lhcb1*3 promoterand of DNA fragments used in EMSA analysis along with indications ofnucleotide modifications that reduce CCA1 binding; in the probesequences (WT1, m1, m2, m3, m4, and WT2) dashes indicate thosenucleotides that are identical to the A2 probe while dots denote gapsintroduced to optimize the alignment of conserved sequence elements;

[0011]FIG. 2 shows the complete nucleic acid sequence of CCA1, thegenomic clone corresponding to the CCA1 cDNA along with the deducedamino acid sequence of the coding portions of the gene;

[0012]FIG. 3 shows the predicted amino acid sequence of CCA1 from aminoacid residue 24 to 75 compared to the repeat sequences of various Mybproteins from animals, plants, and yeast;

[0013]FIG. 4 shows results of low-stringency hybridization ofArabidopsis DNA with a CCA1 probe;

[0014]FIG. 5 shows diagrams of the constructs used for expression ofCCA1 polypeptides in E. coli;

[0015]FIG. 6 shows EMSA results for DNA binding activities ofpolypeptides produced from the constructs of FIG. 5; each reactionincluded 0.3 ng of ³²P-labeled A2 DNA probe and 1 μg poly(dI-dC); lanes1-10, 15, and 16 each contain 1 μg of protein for E. coli either induced(+) or not induced (−) with IPTG; lanes 11 and 13 each contain 50 ng ofpurified GST-CCA1 fusion protein (Factor Xa [−]); and lanes 12 and 14each contain 50 ng of purified CCA1 protein released from the fusionprotein by Factor Xa cleavage (Factor Xa[+]);

[0016]FIG. 7 shows on the left the results of EMSA with the A2 fragmentand the amount of proteins and poly (dI-dC) shown above each lane; F.,free probe; B, CA-1 protein-DNA complex; B1 and B2, CCA1 protein-DNAcomplexes; on the right is shown the sequencing gel of the cleaved DNArecovered from the phenanthroline-copper reaction with S lanesrepresenting the G+A chemical sequencing reaction and with the actualsequence of the protected region spelled out;

[0017]FIG. 8 shows gels of the effects of DNA modification on CCA1binding where partially methylated (gels I and II) or depurinated (gelsIII and IV) A2 probe was labeled at the 3′ end of either the codingstrand (I and III) or the noncoding strand (II and IV) were incubatedwith CCA1; the free DNA (F), the protein-bound DNA (B) and DNA notincubated with protein (C) were cleaved with piperidine and separated onsequencing gels; arrows mark the positions at which modification of DNAinterferes with CCA1-DNA binding;

[0018]FIG. 9a shows the results of a competition experiment with CCA1;

[0019]FIG. 9b shows the results of a competition experiment comparableto FIG. 9a but using plant extract CA-1 instead of E. coli expressedCCA1;

[0020]FIG. 10 shows diagrams of constructs for expression of antisenseCCA1 in transgenic plants; 35S is the promoter of the cauliflower mosaicvirus; NOS the transcription termination sequence of the nos gene whilearrows indicate the sense direction of the CCA1 gene with nucleotidepositions numbered;

[0021]FIG. 11 shows results of RNase protection assays for Lhcb1*3 andrbcS-1A RNA; dark grown seedlings were given no light (D), 2 min R (R)or 2 min R followed by 10 min FR (F) 4 hours before harvesting; WT, wildtype; Line 21 transformed with CCA1-AS21 construct; all other linestransformed with CCA1 -AS 1.4; ubq3 gene used as internal control;

[0022]FIG. 12 shows the induction of CCA1 RNA in etiolated seedlingswhich were grown for six days in the dark and then transferred tocontinuous light; RNA samples were taken either immediately before thetransfer (0) or at the specified time after transfer and analyzed on gelblots;

[0023]FIG. 13a shows the circadian concentrations of CCA1 protein in awild type plant along with the concomitant response of Lhcb1*3 RNA;

[0024]FIG. 13b shows the circadian concentrations of CCA1 protein andLhcb1*3 RNA in a transgenic plant where CCA1 is constitutivelyexpressed;

[0025]FIG. 14 shows a plot of CCA1 protein level versus hypocotyl lengthin a number of transgenic plant lines that were transformed with a CCA1nucleic acid sequence according to the present invention; and

[0026]FIG. 15 shows a plot of CCA1 protein level versus bolting time (indays from seed germination) in a number of transgenic plant lines thatwere transformed with a CCA1 nucleic acid sequence according to thepresent invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] The following description is provided to enable any personskilled in the art to make and use the invention and sets forth the bestmodes contemplated by the inventors of carrying out their invention.Various modifications, however, will remain readily apparent to thoseskilled in the art, since the general principles of the presentinvention have been defined herein specifically to provide the nucleicacid sequence, amino acid sequence and cloned protein of aphytochrome-regulated transcription factor that shows unexpected effectson development and flowering of plants.

[0028] Plant Materials and Growth Conditions

[0029]Arabidopsis thaliana ecotype Columbia was used in all experimentsexcept for ecotype Nossen (No-O) which was used in transformationexperiments with the antisense CCA1_constructs. The medium used forplant growth (MS2S medium) contained 1X MS salts (GIBCO BRL), 0.05% Mes,pH 5.7, 0.8% Phytagar (GIBCO BRL) and 2% sucrose. Light-grown plantswere maintained at 24° C. in a growth chamber with light intensity of150 μE m⁻² sec⁻¹ Growth and light treatments of etiolated seedlings forphytochrome experiments were as described previously (Brusslan, J. A.,and Tobin, E. M., Proc. Natl. Acad. Sci. USA 89:7791-95 (1992)). Whiteonions used in nuclear localization experiments were purchased from alocal supermarket.

[0030] Isolation and Sequence Characterization of CCA1 cDNA and GenomicClones

[0031] Poly(A) RNA was isolated from leaves of Arabidopsis grown for 3weeks on soil in continuous white light. A directional cDNA expressionlibrary was constructed in λgt22A using the SuperScript Lambda system(Bethesda Research Laboratory, Bethesda, Md.). The library was screenedessentially as described by Singh, H., Clerc, R. G., and LeBowitz, J.H., BioTechniques 7:252-61 (1989), except that NEB buffer (25 mMHepes-NaOH, pH 7.2, 40 mM KCl, 0.1 mM EDTA, 5 mM β-mercaptoethanol, 10%glycerol, Sun et al. 1993) was used as the binding buffer, and thewashing solution was supplemented with 0.25% non-fat milk and 0.1%Triton X-100.

[0032] An Arabidopsis λ cDNA expression library was screened with theradiolabeled A2 fragment of the Lhcb1*3 promoter because this fragmenthad been previously shown to interfere with CA-1 binding activity inplant extracts. Approximately 640,000 unamplified recombinant phageplaques were screened in the first round using double-stranded A2 DNAprobe (A2, FIG. 1). The positive plaques from the initial screening wererescreened using both the A2 probe and a mutant probe (ml probe, FIG. 1)that is known to poorly bind to the CA-1 activity (Sun et al. 1993). Twophage clones (clones 21 and 24) that bound only to the A2 and not to theml probe were isolated as individual plaques. The cDNA inserts weresubcloned into the SalI and NotI restriction sites of pGEM11Zf(−)(Promega, Madison, Wis.). Sequence analysis showed that the two clonesoverlapped by 470 nucleotides and were partial cDNAs derived from thesame mRNA. Clone 24 included a polyadenylated tail of 15 bases and,therefore, encompassed the entire 3′ region of the mRNA. The 5′ end ofthe mRNA was determined by primer, extension analysis as described byAusubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J.G., Smith, J. A., and Struhl, K., eds., Current Protocols in MolecularBiology (New York: Greene Publishing Associates and Wiley-Interscience)(1987). The sequence of the oligonucleotide primer corresponded tonucleotides +42 to +22 of CCA1 cDNA clone 21. Fifty fmoles of³²P-labeled primer was annealed to 45 μg of total RNA. The primer wasextended using 9.5 units of Avian Myeloblastosis Virus (AMY) reversetranscriptase (Promega) at 37° C. for 1 hr. Dideoxy sequencing reactionswere performed using the same ³²P-labeled oligonucleotide primer andCCA1 clone 21 plasmid DNA. This demonstrated that clone 21 included thecomplete 5′ region of the transcript. A full-length cDNA clone,designated clone 25, was constructed by joining the 5′ and 3′ fragmentsof clones 21 and 24, respectively, at the unique PstI site in theoverlapping region. The 5′ end of the cDNA clone 24 was removed as aSalI-PstI fragment and replaced with that of clone 21. Sequencing theregion spanning the PstI junction of clone 25 confirmed thereconstitution of wild-type sequence. The sequence of clone 25 ispresented as SEQ ID NO:3.

[0033] A genomic clone corresponding to the CCA1 cDNA was isolated byscreening a genomic library of Arabidopsis thaliana ecotype Columbia inλGEM1 1 (Promega, Madison, Wis.), using the SstI-NotI fragment of CCA1cDNA clone 24 (corresponding to nucleotides 950-2254 of the full-lengthcDNA). The sequences of the CCA1 cDNA and overlapping fragments of thegenomic clone were determined by the dideoxy chain termination methodusing a Sequenase kit (United States Biochemicals, Cleveland, Ohio) anddouble-stranded plasmid DNA. Both strands of the cDNA and genomic DNAwere completely sequenced.

[0034] The sequence of complete gene is shown in FIG. 2 (also SEQ IDNO:1) along with the predicted amino acid sequence for the CCA1 protein.The gene sequence includes seven introns (the first in the 5′ noncodingregion from nucleotide 190 to 273; the others from nucleotides 361 to438, 551-638, 701-1179, 1375-1461, 1636-1718, and 2601-2670), 237nucleotides of 5′ untranslated sequence, and 193 nucleotides of 3′untranslated sequence. The 1824-nucleotide open reading frame (ORF)encodes a protein of 608 amino acids with a calculated molecular weightof 66,970 and an isoelectric point of 5.6. An ORF of 24 nucleotides ispresent in the 5′ untranslated region of the transcript and is in phasewith the main ORF. Such ORFs have been shown in several cases to beinvolved in translational regulation of gene expression (Lohmer, S.,Maddaloni, M., Motto, M., Salamini, F., and Thompson, R. D., Plant Cell5:65-73 (1993); Hinnebusch, A. G., Trends in Biochem.Sci. 19:409-1.4(1994)), and have also been found in other plant transcription factorgenes (Singh, K., Dennis, E. S., Ellis, J. G., Llewellyn, D. J.,Tokuhisa, J. G., Wahleithner, J. A., and Peacock, W. J., Plant Cell2:891-903 (1990); Ruberti, I., Sessa, G., Lucchetti, S., and Morelli,G., EMBO J. 10:1787-91 (1991); Carabelli, M., Sessa, G., Baima, S.,Morelli, G., and Ruberti, I., Plant J. 4:469-79 (1993); Lohmer et al.,1993).

[0035] Sequence Analysis and Data Base Searching

[0036] The protein and DNA sequences were analyzed using the MacVectorsoftware (IBI, New Haven, Conn.) and the Genetics Computer Group(Madison, Wis.) software package. The GenBank data base was searchedwith the amino acid sequence of CCA1by using the BLAST (Altschul, S. F.,Gish, W., Miller, W., Myers, B. W., and Lipman, D. J., J. Mol. Biol.215:403-10 (1990)) and FASTA programs (Pearson, W. R. and Lipman, D. J.Proc. Natl. Acad. Sci. USA 85:2444-48 (1988)) on the National Center forBiotechnology Information (NCBI) on-line service. Sequence alignment wasassembled manually based on the results of data base searches. Genomicsequences of light-harvesting complex apoprotein (Lhc) genes and smallsubunit of ribulose bisphosphate carboxylase/oxygenase (rbcS) genes wereretrieved from the Genbank data base using text search on the NCBI worldwide web site. The presence of AATCT sequences in the promoter regionsof the genes was detected using the FASTA program and further analyzedvisually.

[0037] The predicted amino acid sequence of the CCA1 protein has a basicregion at the N terminus (K-13 to K-107). Within this region is asequence similar to the repeat sequence highly conserved in Myb-relatedproteins. FIG. 3 shows the predicted amino acid sequence of CCA1 fromamino acid residue 23 to 75 compared to the repeat sequences of variousMyb proteins from animals, plants, and yeast. Within this sequence,there is a limited amino acid identity (16 of 52; 31%) and substantialsimilarity (29 of 52; 56%) when compared to the third repeat of humanc-Myb. The sequence identity includes two of the three conservedtryptophans present in most Myb proteins. The conserved residues alsoinclude seven of the 11 residues that are known to be important forforming the hydrophobic core and maintaining the three-dimensionalstructure of the Myb repeat, which forms a helix-turn-helix structure(Ogata, K., Hojo, H., Aimoto, S., Nakai, T., Nakamura, H., Sarai, A.,Ishii, S., and Nishimura, Y., Proc. Natl. Acad. Sci. USA 89:6428-32(1992)). However, the amino acid residues of human Myb that actuallycontact the DNA bases are not conserved in CCA1 (N-183 in hMyb, S inCCA1; K-182 versus R; N-186 versus Q; N-179 versus V) (Ogata, K.,Morikawa, S., Nakamura, H., Sekikawa, A., Inoue, T., Kanai H., Sarai,A., Ishii, S., and Nishimura, Y., Cell 79:639-48 (1994)). In contrast tomost other Myb proteins that have been characterized, this region is notrepeated in the CCA1 protein. No other significant homology to anyprotein in the data base was found.

[0038] DNA and RNA Gel Blot Analyses

[0039] Genomic DNA isolation and DNA gel blotting were performed asdescribed by Brusslan et al. (1993). Membranes were hybridized withP-labeled CCA1 cDNA fragments under high stringency conditions (finalwashes were at 65° C. in 0.1% SSC, [1X SSC is 0.15 M NaCl, 0.015 Msodium citrate] 0.1% SDS) and then stripped and reprobed underlow-stringency conditions (hybridization at 32° C. in buffer containing50% formamide, 0.25 M NaHPO₄, pH 7.2, 0.75 M NaCl, 7% [w/v] SDS, and 1mM EDTA and final washes at 45° C. in 2X SSC, 0.1% SDS). Total RNA wasextracted from Arabidopsis seedlings as described by Brusslan and Tobin(1992). Total RNA was separated on a 1% agarose gel containingformaldehyde and blotted onto ZetaProbe membrane (Bio-Rad, Richmond,Calif.) following the manufacturer's instructions. RNA probes weresynthesized by in vitro transcription using linearized plasmid DNA. CCA1RNA probe was synthesized from CCA1 clone 24. To make the ubq10 RNAprobe, a fragment of the 3′ untranslated region of the ubq10 gene(Callis, J., Carpenter, T., Sun, S. W., and Vierstra, R. D., Genetics139:921-39 (1995)) was amplified by polymerase chain reaction (PCR)using the primers: 5′-CTGTTATGCTTAAGAAGTTCAATGT-3′ and5′-CCACCCTCGAGTAGAACACTTATTCAT-3′.

[0040] The amplified fragment was digested with HindIII and XhoI andcloned into pGEM-11Zf(−). This plasmid DNA was digested with HindIII andused as template for synthesis of ubq10 RNA probe. The Lhcb1*3 RNA probewas made as described by Brusslan and Tobin (1992). The membrane blotwas hybridized overnight with the RNA probes in buffer containing 50%formamide, 0.3 M NaCl, 0.05 M NaHPO₄, pH 6.5, 1 mM EDTA, 1% SDS, 0.1%Ficoll (type 400), 0.1% polyvinylpyrrolidone, 0.1% BSA, 0.5 mg/mL yeasttRNA and 0.5 mg/mL herring sperm DNA. Hybridization of Lhcb1*3 ubq10 andCCA1 probes was performed at 55° C., 52° C. and 58° C., respectively.Final washes were performed at 65° C. in 0.1X SSC, 0.1% SDS. Afterhybridization with Lhcb1*3 and ubq10 probes, the blot was stripped byboiling in 0.1X SSC, 0.1% SDS, then hybridized with the CCA1 probe. Theblots were imaged and quantified using a Phosphorlmager (MolecularDynamics, Sunnyvale, Calif.). The measurement of the signal for eachprobe was adjusted for the uridine content of the probe and the exposuretime, and the Lhcb1*3 and CCA1 signals were normalized to the ubq10signal.

[0041] We tested whether CCA1 is a member of a gene family inArabidopsis by genomic DNA gel blot analysis. The results oflow-stringency hybridization of Arabidopsis DNA with a CCA1 probe areshown in FIG. 4. There was a single band of hybridization in the lanesthat were digested with EcoRI, SstI, and PstI, which have no cleavagesite in the probe fragment. There were two bands in the lane that wasdigested with HindIII, which has a cleavage site in the probe 188 bpfrom one end. An identical pattern was seen when the blot was hybridizedwith the same probe under the high-stringency conditions. The DNA gelblots were also hybridized under both low- and high-stringencyconditions with a probe consisting of nucleotides 267-949 of the CCA1cDNA, which includes the region of similarity to the Myb repeat. Thisprobe gave no evidence for any additional closely related sequences. Weconclude that although the CCA1 gene includes a small region with aminoacid sequence homology to the Myb repeat, there are no genes that areclosely related to CCA1 in the Arabidopsis genome.

[0042] Partial purification of Arabidopsis CA-1 protein, A2 probelabeling, and the electrophoretic mobility shift assays (EMSAs) werecarried out as described by Sun et al. (1993). Competitor DNA fragmentswere prepared by annealing synthetic oligonucleotides. Competition EMSAexperiments were performed by adding partially purified plant CA-1protein or affinity-purified CCA1 polypeptide expressed from pXCA-24 inE. coli to the DNA binding reaction mixture containing A2 probe andspecified amounts of competitor DNA fragments. The dried gels wereimaged and quantified using a Phosphorlmager.

[0043] The fact that the two cDNA clones isolated in the initialfilter-binding screening each contained the sequence similar to the Mybrepeat suggested that this region was necessary for DNA binding. Wetested this possibility and further characterized the CCA1 protein byexpressing the polypeptides encoded by various fragments of the CCA1cDNA. The CCA1 cDNA clones were fused to the coding sequence forglutathione 5-transferase (GST) and used to produce the polypeptides inEscherichia coli. These constructs are diagrammed in FIG. 5. Thepolypeptides produced were tested for their ability to bind to the A2fragment of the Lhcb1*3 promoter by electrophoretic gel mobility shiftassays (EMSA). The polypeptides corresponding to the pXCA-23 and pXCA-24constructs were produced as isopropyl β-d-thiogalactopyranoside(IPTG)-inducible GST-fusion proteins, and were also tested as purifiedproteins after cleavage from GST. Those corresponding to the cDNA clonesCCA1-21 and CCA1-25 (pXCA-21 and pXCA-25, respectively) contained stopcodons in the 5′ untranslated region of the cDNA and, thus, were notproduced as fusion proteins. FIG. 6 shows an EMSA using either E. coliextracts (lanes 1 to 10, 15 and 16) or with purified proteins before(lanes 11 and 13) and after (lanes 12 and 14) cleavage from GST. DNAbinding activities induced by IPTG were observed for proteins producedfrom constructs pXCA-21, pXCA-24, and pXCA-25, but binding activitycould not be detected for the protein produced from construct pXCA-23,which lacked the N-terminal 82 amino acids In conjunction with thefinding that the N-terminal 11 amino acids are not necessary for binding(construct pXCA-24), these experiments demonstrate that the sequencecontaining amino acid residues 11-82 of CCA1, which includes the regionwith similarity to the Myb DNA binding domain (amino acids 24-75)., isessential for the DNA binding activity of the CCA1 protein. Therefore,homologous CCA1 proteins from other plant species will share this highlyconserved binding domain most likely with 85% or higher homology. Theasterisk in the figure marks a nonspecific DNA binding activity. Thearrow and triangles denote the positions of the major protein-DNAcomplexes formed by the GST-CCA1 fusion protein and the non-fusion CCA1polypeptides, respectively. Lanes 15 and 16 are longer exposures oflanes 5 and 6.

[0044] Protein Expression in Escherichia coli and Purification ofGST-CCA1 Proteins

[0045] The constructs diagrammed in FIG. 5 were made by cloning the CCA1cDNA fragments into pGEX-3X (Pharmacia) using polymerase chain reaction(PCR)-aided cloning with the following 5′ primers:5′-GGCCGGGATCCAATTCGTCGACCCACGCG-3′ for pXCA-21, pXCA-24, pXCA-25 and5′-TAAAGGGATCCATATGGGTCAAGCGCTAG-3′ for pXCA-23. A 3′ primer(5′-ATAGAATTCTCGAGCTTATGCATGCGG-3′) was used for pXCA-21, pXCA-24,pXCA-25 and pXCA-23. The appropriate plasmid DNA (0.5 μg) was amplifiedfor 10 cycles and the PCR products were digested with EcoRI and BamHI.The cDNAs of clones 21, 24, and 25 and the 483 to 2254-nucleotide regionwere cloned into pGEX-3X yielded pXCA-21, pXCA-24, pXCA-25 and pXCA-23,respectively. Sequencing of the junction region between the glutathioneS-transferase (GST) gene and cDNA confirmed the construction of atranslational fusion in pXCA-24 and pXCA-23.

[0046] The plasmid constructs were transformed into E. Coli BL21 (DE3).Protein expression, purification of GST-CCA1 fusion proteins usingglutathione agarose, and purification of CCA1 polypeptides by cleavageof matrix-bound GST fusion protein with Factor Xa were performedfollowing the procedure of Ausubel et al. (1987). Protein concentrationswere determined by the Bradford assay (Bio-Rad, Richmond, Calif.) usingBSA as standard.

[0047] Phenanthroline-Copper Footprinting

[0048] The A2 fragment was labeled with ³²P at the 3′ end of the sensestrand by end filling (Sun et al., 1993). Footprint experiments werecarried out as described by Kuwabara, M. D., and Sigman, D. S.,Biochemistry 26:7234-38 (1987). The EMSA reactions were scaled upfivefold; 10⁶ cpm of probe and specified amounts of protein andpoly(dI-dC) were used in each reaction. After electrophoresis, the gelwas treated with phenanthroline-copper, then exposed wet to x-ray filmfor 40 min. The bands representing free DNA and protein-DNA complexeswere excised from the gel. DNA was eluted from the gel slices, recoveredby ethanol precipitation, and loaded on an 8% polyacrylamide-ureasequencing gel. The G+A chemical cleavage sequencing reaction wasperformed as described by Maxam, A. M., and Gilbert, W., Methods Enzymol65:499-580 (1980).

[0049] Partial methylation and depurination of the A2 DNA probe wasperformed following the procedure of DNA chemical sequencing (Maxam andGilbert, 1980). Five ng (10⁵ cpm) of modified DNA probe was incubatedwith 0.8 μg of affinity purified CCA1 protein in 50 μL NEB buffercontaining 5 μg poly(dI-dC) and 10 μg BSA. The protein-bound and freeDNA were separated by filtering the mixture through a nitrocellulosemembrane (Ausubel et al., 1987). Free and bound fractions of DNA wererecovered, and cleaved with piperidine following the DNA chemicalsequencing procedure. An aliquot of probes not incubated with proteinwas also cleaved with piperidine as a control. Equal amounts ofradioactivity from each sample were used on an 8% polyacrylamide-ureasequencing gel.

[0050] To compare the binding characteristics of the CCA1 protein andCA-1 activity from the plants, we carried out footprint analyses andbinding competition experiments using the A2 fragment of Lhcb1*3promoter as a probe. The results of 1,10-phenanthroline-copperfootprinting are shown in FIG. 7. At left is the EMSA that was performedto resolve the free probe and the DNA-protein complexes. Cleaved DNA wasrecovered from each band after treatment of the gel withphenanthroline-copper and resolved on the sequencing gel shown at right.With increasing amounts of the CCA1 protein purified from E. coli,complexes (B1 and B2) of different mobilities could be observed. Thenucleotides protected from cleavage in each of the complexes can be seenon the sequencing gel on the right. In complex B1, the −92 to −105region was protected, and in complex B2, regions from −92 to −105 andfrom −111 to −122 were protected. This result suggests that the twocomplexes of different mobilities are a result of the presence of twoseparate binding sites on this fragment, and that the −92 to −105 regionis the higher affinity binding site for CCA1. A nearly perfect repeatedsequence of AAA^(A)/_(C)AATCTA occurs in each of these footprintedregions.

[0051] The CA-1 protein-DNA complex obtained with the plant cell extract(FIG. 7, lane 4) showed protected nucleotides in the region from −94 to−105, and a second experiment confirmed these boundaries, demonstratingthat CA-1 (from plant extracts) and CCA1 (from the clone expressed in E.coli) bind to the same region of the Lhcb1*3 promoter.

[0052]FIG. 8 show the results of methylation interference anddepurination interference experiments performed with the CCA1 protein.The figure shows the interfering nucleotides on sequencing gels, andtheir position on the A2 fragment of the promoter is shown in FIG. 1,along with the results of footprinting experiments. Interference withthe protein-DNA binding by the modification of a base residue ismanifested by increased intensity in the lane with the free DNA fractionand reduced intensity in the lane of protein-bound DNA compared to thelane of control DNA that was not incubated with protein. Bothmethylation and depurination interference assays identified the samenucleotides, and showed that nucleotides within both nearly perfectrepeats (AA^(A)/_(C)AATCTA) interact directly with the CCA1 protein. InFIG. 1 thick and thin lines 12 and 14 show the regions protected by CCA1and CA-1, respectively, in the footprint assay. Asterisks in the figureindicate nucleotides that interfere with CCA1-DNA binding whenmethylated; boldface indicates the nucleotides that interfere withbinding when depurinated.

[0053]FIG. 1 also summarizes the results of the phenanthroline-copperfootprinting. We used unlabeled competitor DNAs in the EMSA to comparebinding specificities of the CCA1 protein produced in E. coli and theCA-1 activity from the plant extracts. The wild-type and mutant promoterfragments used as competitors are shown at the bottom of FIG. 1. Arepresentative result of such experiments is shown in FIG. 9a for CCA1and FIG. 9b for CA-1. The binding of the E. coli-produced CCA1 proteinto the probe was efficiently competed for by either a fragment of the A2probe that contained the repeated sequence or by a promoter fragment(WT2) of another closely related Lhcb gene (Lhcb1*1, originally calledAB165; Leutwiler, L. S., Meyerowitz, E. M., and Tobin, E. M., Nucl.Acids Res. 14:4051-64 (1986)) that contains one copy of this sequence(AAAAATCT). The m3 fragment, which had altered nucleotides in thedownstream repeat region, was a less effective competitor than was thewild-type (WT1); m1, m2, and m4 fragments, which had alterations in bothrepeats, showed the least competition.

[0054] When plant extracts were used, all the fragments showed somedegree of competition, which is likely in part to be the result of lowamounts of the CA-1 protein in plant extracts. The results are notdirectly comparable to those with the purified CCA1 protein because theabsolute amounts of the specific binding proteins are not known.Nonetheless, it can be seen that the m2 fragment served as a bettercompetitor for CA-1 than did the ml fragment, whereas the opposite wasfound with CCA1. Even more striking are the contrasting results with them4 competitor DNA. This fragment, in which the C residues of both TCTmotifs in the two repeats were changed, was even more effective than wasthe wild type in competing for the CA-1 activity, whereas it was not aparticularly good competitor for CCA1. Thus, although both activitiesinteract with the AAAAATCT sequence, there are differences in theimportance of individual nucleotides in this sequence for the binding ofCA-1 and CCA1.

[0055] The CCA1 protein interacted with two closely spaced binding siteswith nearly perfect 10-bp repeated sequences (AAA^(A)/^(C)AATCTA) in theLhcb1*3 promoter. Previous results (Sun et al., 1993) and the results ofthe phenanthroline-copper footprinting (FIG. 7) show that the CA-1-activity could protect the same nucleic acid sequence as CCA1. Thereare, however, some differences in the relative importance of specificnucleotides for the binding of the two activities. The binding of CCA1was more affected by alteration in the TCT sequence than by alterationsin the AAAAA, whereas the opposite was observed with the plant extractactivity (cf. m3 and m4, FIG. 9). It is possible that the differencesobserved are due to differences in modifications of the protein in E.coli and plants or that the CA-1 activity in the plant extracts might beassociated with an additional protein or proteins which alter thebinding characteristics. It is also possible that CA-1 and CCA1 areactually the products of two different genes, or the result ofalternative splicing, in which case they may compete for the samebinding sites.

[0056] Nuclear Localization

[0057] Onion epidermal peels were transformed by biolistictransformation and analyzed for GUS activity, and nuclei localizationwas as described in Varagona, M. J., Schmidt, R. J., and Raikhiel, N.V., Plant Cell 4:1213-27 (1992). Histochemical staining was visualizedusing a Zeiss Axiophot microscope and photographed using KodakEktachrome (Elite Series) ASA 400 film.

[0058] Transient expression assay in onion epidermal cells testedwhether the product of the CCA1 gene was localized to nuclei, as wouldbe expected for a transcription factor. The uidA gene, which encodes,β-glucuronidase (GUS), was fused in frame to the coding sequence of CCA1so that GUS activity could be used to localize the compartmentation ofthe CCA1 protein. An XbaI site and a BamHI site were introduced intoCCA1 by PCR amplification of cDNA clone 25 using the 5′ primer

(5′-GAAGTTGTCTAGAGGAGCTAAGTG-3′)

[0059] and 3′ primer (5′-ATGTGGATCCTTGAGTTTCCAACCGC-3′) (mismatches areunderlined). The resulting PCR product was digested with XbaI and BamHIand inserted in pB1221 (Clontech, Palo Alto, Calif.), yieldingp35S-CCA1-GUS. This construct contains CCA1_coding sequence as a 1828-bpXbaI-BamHI fragment inserted between the cauliflower mosaic virus 35Spromoter and the uidA gene. pMF::GUS and pMF::B::GUS were obtained fromN. Raikhel (Michigan State University, East Lansing, Mich.);construction of these plasmids is described in Varagona et al. (1992).This transient assay should result in the expression of GUS activity inindividual cells into which the DNA is effectively introduced. When aCaMV 35S::uidA construct (pMF::GUS) was used in this assay, GUS activitywas detected throughout the cytoplasm. When a construct (pMF::B::GUS)with the opaque2 gene, which encodes a well characterized transcriptionfactor from maize, fused to the uidA gene was used, GUS activity wasdetected specifically in nuclei. Similarly, specific nuclearlocalization was found for the CCA1-GUS (p35S-CCA1-GUS) fusion protein.These results show that the CCA1 protein is targeted to nuclei and areconsistent with the function of the CCA1 protein as a transcriptionfactor.

[0060] Plant Transformation Antisense

[0061] It has previously been shown that the promoter region to whichthe CA-1 activity binds is essential for phytochrome regulation of theLhcb1*3 gene (Kenigsbuch and Tobin, 1995). Therefore, if the product ofthe CCA1 gene interacts with this promoter in vivo, it might be expectedto affect the phytochrome induction of Lhcb1*3 expression. We addressedthis possibility by transforming Arabidopsis with portions of the CCA1gene in an antisense orientation driven by the constitutive cauliflowermosaic virus 35S promoter (see FIG. 10). The SstI-NotI fragments of CCA1clones 21 and 24 in pGEM11Zf(−) were cloned into pBluescript KS(Stratagene, La Jolla, Calif.) at the corresponding sites. The resultingplasmids were digested with BamHI and SstI, and the cDNA fragments werecloned into the BamHI and SstI sites of binary vector pB1121 (Clontech),replacing the uidA gene coding sequence. The binary vectors weretransformed into Agrobacterium tumefaciens A2260. Arabidopsis ecotypeNo-O plants were transformed with the above constructs using theAgrobacterium-mediated root transformation procedure described byValvekens, D., Van Montagu, M., and Van Lijsebetten, M., Proc. Natl.Acad. Sci. USA 85:5536-40 (1988).

[0062] For each independent transgenic line, T2 seeds homozygous for theT-DNA insertion were selected by analysis of the segregation ofkanamycin resistance, and seedlings from these homozygous seeds weretested for the phytochrome responsiveness of both the endogenous Lhcb1*3gene and another phytochrome regulated gene, the rbcS-IA gene. FIG. 11shows that in five of these seven lines, the level of Lhcb1*3 mRNA afterthe red treatment was substantially lower than that of the wild-type.However, no substantial effect of the antisense construct was seen forrbcS-IA gene expression in the same lines. The mRNA levels for Lhcb1*3and rbcS-IA were normalized to a ubiquitin RNA (ubq3; cf Brusslan andTobin, 1992), and the relative expression levels of these two genes forall the lines and treatments are shown below the autoradiogram. Theincrease of Lhcb1*3 RNA in response to R was reduced in lines 4, 14, 17,21, and 34, ranging from 37-53% that of the wild type, but the inductionof the rbcS-IA RNA was not comparably affected. The fact that none ofthe lines we recovered showed strong suppression of CCA1 RNA suggeststhe possibility that complete loss of function might be highlydeleterious. It is also notable that the antisense construct did notaffect the mRNA levels in plants that had been given no light treatmentor had been given FR following the R treatment. No obvious visiblephenotype was apparent in the antisense lines.

[0063] We also used the T3 generation of four of the lines to testwhether the reduction in Lhcb1*3 RNA correlated with a reduction inlevels of CCA1 RNA. In this generation, there was a smaller effect ofthe antisense construct. It is not unusual for antisense effects to belost or diminished in subsequent generations. For example, Chamnongpol,S., Willekens, H., Langebartels, C. Van Montagu, M. Inze, D. And VanCamp, W., Plant J. 10:491-503 (1996) found that seven of eight linesexpressing an antisense construct for a catalase gene lost the catalasesuppression phenotype in their progeny. The R induction of Lhcb1*3 RNAranged from 68 to 86% of the wild-type in the T3 seedlings of these fourlines, and the levels of CCA1-RNA were 68 to 75% that of the wild-typeplants. Although the effect of the antisense constructs wassubstantially reduced in this generation, the reduction of CCA1 RNA wasaccompanied by a similar decrease in the induction of Lhcb1*3 RNA by R.Our results demonstrate that the CCA1 protein can have a specific effecton the phytochrome induction of expression of the endogenous Lhcb1*3gene in vivo, and strongly indicate that this protein is a part of thenormal transduction pathway for the phytochrome response of this gene.

[0064] Plant Transformation Constitutive Expression

[0065] As demonstrated above, CCA1 is a transcription factor that isintimately involved in the phytochrome-induced regulation of Lhcb1*3 andthe corresponding light harvesting chlorophyll binding protein. Somewhatparadoxically CCA1 may be itself phytochrome regulated. FIG. 12 showsthe time course of production of CCA1 and Lhcb1*3 RNAs when dark-grownseedlings are transferred into light. CCA1 is induced within 1 hr andpeaks at before 2 hr, thereafter decaying away. Lhcb1*3 begins to appearfollowing the peak in CCA1 and continues at a high level as long as theplants remain in the light. This transient CCA1 response might suggestinvolvement of CCA1 in more complex events.

[0066] One way of exploring the overall effects of the phytochromecontrol of CCA1 is to remove it from this phytochrome control andascertain what, if any effects, there are on plant growth anddevelopment. This can be achieved by reversing the direction of the CCA1sequence inserted into the constructs shown in FIG. 10. Now instead ofproducing an antisense message, the CCA1 gene behind the 35S cauliflowermosaic promoter results in constitutive production of the CCA1 protein.FIG. 13a shows the circadian variations in wild-type expression of CCA1.Here the plants have been grown under a normal light/dark regime andthen transferred into continuous light. The endogenous circadian rhythmof CCA1 production continues even under constant light. This rhythmclosely matches the original light/dark period as is indicated by thetime line. Note that the CCA1 is fully expressed at the start of thelight period (ZT 1). This production decays before the end of the lightperiod so that by the start of the dark period (ZT 12) CCA1 productionis essentially absent—even though the plants remain under constantillumination. CCA1 production leads the beginning of the next lightperiod (ZT 25) being apparent by ZT 19. This circadian rhythm wasoriginally entrained to light/dark periods but clearly continues inplants kept in constant light. Lhcb1*3 RNA tracks CCA1 but decays moreslowly so that its level reaches a minimum during the dark period somehours after minimal CCA1 levels have been reached. This difference inphase between CCA1 and Lhcb1*3 RNA results from a lag between CCA1protein synthesis and RNA synthesis and slow turnover of Lhcb1*3 RNA.However, it is possible that this lag is actually part of the “clock”mechanism by which the plant actually maintains it circadian rhythm.

[0067]FIG. 13b shows that when CCA1 is constitutively expressed in atransgenic plant not only does CCA1 levels remain constant(as expected)but Lhcb1*3 RNA levels also remain constant, thereby damping orobscuring the entire circadian rhythm of Lhcb1*3. In the presence oflight the expression pattern of CCA1 now controls the expression of.Lhcb1*3. However, if CCA1 is actually part of the “clock” mechanism,overall results may be more profound.

[0068] The real question, then, is whether this apparent damping of thecircadian rhythm affects only the level of Lhcb1*3 transcription orrepresents a more widespread influence on the “clock” that controlsplant development and whether such an effect is exhibited under normallight/dark regimes as opposed to constant illumination. Generally thephenotype of the 35S-CCA1 plants is normal. One of the only detectablemorphological effects appears to be a relationship between hypocotyllength measured at six days and CCA1 level in a given plant. CCA1 levelscan vary considerably from one transgenic line to another. FIG. 14 showsa regression analysis of hypocotyl length versus CCA1 level for 14different transgenic lines. There is a strong correlation (r=0.73)between increased hypocotyl length and increased levels of CCA1indicating that longer hypocotyls results from CCA1overexpression-certainly not a result expected from a transcriptionfactor that controls Lhcb1*3 RNA. Normally, hypocotyls are shorter inbright light than in dark-grown plants. Perhaps the constitutive CCA1expression is interfering with the plant's perception of light versusdark. This does suggest that CCA1 effects go beyond Light harvestingchlorophyll binding protein.

[0069] Much more exciting and totally unexpected is the effect of CCA1level on days to flowering of Arabidopsis. Normally the plants areinfluenced both by day length and by days of growth from seedgermination. Under short day conditions the plants will show arelatively prolonged growth phase before they bolt and begin to flower.However, under long day conditions the plants very quickly transit fromthe vegetative phase to the reproductive phase. That is, if seeds aregerminated late in the season (as the days are growing longer) evensmall seedlings quickly begin to flower completely skipping most of thenormal vegetative growth phase. This same behavior is shown by a largenumber of plants. Most gardeners are well aware of the way thatradishes, spinach and lettuce will begin to flower as the summerapproaches. This behavior is annoying since the palatability of thevegetables is ruined. This behavior is of far greater economicimportance with forage crops, in particular pasture grasses such as ryeor fescue. The main value of these crops is in the food their vegetativestructures provide to domestic animals. When the forage plants begin toflower, their production of vegetative biomass ends and their value as acrop ceases. Until the present invention the only way to deal with thisproblem was the use of traditional plant breeding methods to selectvarieties that were slower to flower. This approach has had some littlesuccess but the selected varieties could be improved. By a further delayin flowering.

[0070]FIG. 15 plots the number of days to flowering (bolting) from seedgermination for a number of different transformed lines that overexpressCCA1. Just as hypocotyl length was related to CCA1 level in FIG. 14, inFIG. 15 bolting time is highly correlated with CCA1 level (r=0.81). AsCCA1 level increases, flowering is delayed so that CCA1 affords thefirst general method for delaying flowering in plants. This is strongevidence that CCA1 is more than just a transcription factor forregulating Lhcb1*3 RNA. Since altering the level of this factorsignificantly delays flowering and apparently damps circadian rhythms itseems likely that this factor is part of the “clock” mechanism and isintimately involved in the regulation of a large number of “timing”related aspects of plant development. Since the DNA-binding portion(amino acids 24-75) of CCA1 is highly conserved it is very likely thatthis protein will be effective in a wide variety of plants and that themethod of the present invention will modify flowering in virtually allplants. Conversely, while other species may have homologous CCA1proteins whose sequences vary from that disclosed herein, the method ofusing those sequences to modify flowering time is identical to thatdisclosed and claimed herein. Alteration of plant development bytransformation of plants with any nucleic acid sequence that translatesto a transcription factor showing significant homology to the keybinding region (amino acids 24-75) of CCA1 is contemplated by thepresent invention.

[0071] Now by constitutively expressing CCA1 it is possible to disruptthe plants built in timing system. With this system disturbed, the plantis much less able to respond to increases in day length as the growingseason progresses. This results in a significant increase in vegetativegrowth and accumulated biomass before a transition to the reproductivestate occurs. In the case of forage crops this translates to a dramaticyield increase as a given planting continues to produce biomass for alonger time. In the case of seed crops (e.g. rape seed a relative ofArabidopsis) a delay of flowering can translate to larger plants and alarger seed yield as long as flowering is not delayed too long into thegrowing season. The first phytochrome-regulated transcription factorprovided by the present invention represents the first known way tomanipulate plant circadian rhythms and hence flowering through geneticengineering. It seems likely that CCA1 will also serve as the key tounlock other aspects of the phytochrome-based timing system in plants.

[0072] Those skilled in the art will appreciate that various adaptationsand modifications of the just-described preferred embodiment can beconfigured without departing from the scope and spirit of the invention.Therefore, it is to be understood that, within the scope of the appendedclaims, the invention may be practiced other than as specificallydescribed herein.

What is claimed is:
 1. A transcription factor comprising a memberselected from the group consisting of: (a) a peptide having an aminoacid sequence of SEQ ID NO:2; (b) a peptide having an amino acidsequence identical to a peptide produced by translation of codingportions of nucleic acid sequence Seq ID NO:1; (c) a peptide having anamino acid sequence identical to a peptide produced by translation ofnucleic acid sequence SEQ NO:3; (d) a peptide having at least 95%sequence homology to peptide (a).
 2. An isolated polynucleotidecomprising a member selected from the group consisting of: (a) apolynucleotide having a sequence identical to SEQ ID NO:1; and (b) apolynucleotide which hybridizes to and which is at least 95%complementary to polynucleotide (a); and (c) a polynucleotide that isexactly complementary to polynucleotide (b).
 3. An isolatedpolynucleotide comprising a member selected from the group consistingof: (a) a polynucleotide having a sequence identical to SEQ ID NO:3; (b)a polynucleotide which hybridizes to and which is at least 95%complementary to polynucleotide (a); and (c) a polynucleotide that isexactly complementary to polynucleotide (b)
 4. A method of alteringplant development comprising transforming a plant with nucleic acidsequence selected from the group consisting of: (a) a polynucleotidehaving a sequence identical to SEQ ID NO:1; (b) a polynucleotide whichhybridizes to and which is at least 95% complementary to polynucleotide(a) (c) a polynucleotide having a sequence identical to SEQ ID NO:3; and(d) a polynucleotide which hybridizes to and which is at least 95%complementary to polynucleotide (c).
 5. A transgenic plant produced bytransforming a plant with a nucleic acid sequence selected from thegroup consisting of: (a) a polynucleotide having a sequence identical toSEQ ID NO:1; (b) a polynucleotide which hybridizes to and which is atleast 95% complementary to polynucleotide (a) (c) a polynucleotidehaving a sequence identical to SEQ ID NO:3; and (d) a polynucleotidewhich hybridizes to and which is at least 95% complementary topolynucleotide (c).
 6. A method of altering plant development comprisingtransforming a plant with a nucleic acid sequence coding for a CCA1protein, said protein having a domain showing at least 85% homology toamino acids 24-75 of SEQ ID NO:2.
 7. A transgenic plant transformed witha nucleic acid sequence coding for a CCA1 protein, said protein having adomain showing at least 85% homology to amino acids 24-75 of SEQ IDNO:2.